Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.     

地衣芽孢杆菌α-淀粉酶基因的克隆和及其启动子功能鉴定

根据已知α-淀粉酶编码基因保守区核苷酸序列,通过PCR和反向PCR技术克隆出Bacillus licheniformisCICIM B0204α-淀粉酶编码基因amyL全长序列及其上下游序列。B.licheniformisCICIM B0204amyL由1539bp组成,其上游180bp为启动子序列,下游160bp为终止子序列;成熟肽由512个氨基酸残基组成,氨基端的29个氨基酸残基为α-淀粉酶的信号肽。通过基因及其氨基酸序列比对发现,amyL及其编码产物与芽孢杆菌来源的α-淀粉酶具有高度相似性。将amyL的结构基因在PT7介导下于大肠杆菌中诱导表达,获得具有α-淀粉酶活性的表达产物。将amyL的启动子序列和信号肽序列与B.licheniformisCICIM B2004的β-甘露聚糖酶结构基因进行读框内重组,在大肠杆菌中获得了β-甘露聚糖酶的分泌表达,重组大肠杆菌表达295U/mL的β-甘露聚糖酶酶活。     

N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Nucleotide sequence of the beta-cyclodextrin glucanotransferase gene of alkalophilic Bacillus sp. strain 1011 and similarity of its amino acid sequence to those of alpha-amylases.

The nucleotide sequence of the gene for cyclodextrin glucanotransferase of alkalophilic Bacillus sp. strain 1011 was determined. The deduced amino acid sequence at the NH2-terminal side of the enzyme showed a high homology with the sequences of alpha-amylase in the three regions which constitutes the active centers of alpha-amylases.     

A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.     

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.     

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. Amino acid replacements at 6 histidine residues reveal a critical position at His-133

A set of 12 Escherichia coli suppressor tRNAs, inserting different amino acids in response to an amber codon, has been used to create rapidly numerous protein variants of a thermostable amylase; by site-directed mutagenesis, amber mutations were first introduced into Bacillus licheniformis alpha-amylase gene at position His35, His133, His247, His293, His406, or His450; genes carrying one or two amber mutations were then expressed in the different suppressor strains, generating over 100 amylase variants with predicted amino acid changes that could be tested for thermostability. Within the detection limits of the assays, amino acid replacements at five histidine positions had no significant effect. In contrast, suppressed variants substituted at residue His133 clearly exhibited modified thermostability and could be either less stable or more stable than the wild-type amylase, depending on the amino acid inserted at this position; comparison of the variants indicates that the hydrophobicity of the substituting residue is an important but not a determinant factor of stabilization. The effect of the most stabilizing and destabilizing amino acid substitutions, His133 to Tyr and to Pro, respectively, were confirmed by introducing the corresponding missense mutations into the gene sequence. The advantages and limits of informational suppression in protein stability studies are discussed as well as structural features involved in the thermostability of B. licheniformis alpha-amylase.     

Processing of the prepropeptide portions of the Bacillus amyloliquefaciens neutral protease fused to Bacillus subtilis alpha-amylase and human growth hormone during secretion in Bacillus subtilis.

A set of nested 3'-terminal deletions of the prepropeptide of the Bacillus amyloliquefaciens neutral protease gene was constructed. Alpha-amylase and human growth hormone were secreted using these truncated genes in Bacillus subtilis. The level of the secreted alpha-amylase varied with the region for the truncated prepropeptide contained in the fusion gene but was independent of its length. Even though length of the prepropeptide varied, the mobilities of secreted alpha-amylases were the same as that of the control alpha-amylase derived from the alpha-amylase clone, pTUB4 (Yamazaki et al., 1983). Analyses of the secreted N-terminal amino acid sequences confirmed that they were all identical to that of the authentic one. Precursor proteins of the alpha-amylase were found in the cell-associated fraction, suggesting that the prepropeptide portion was processed during secretion. On the other hand, the N-terminus of hGH secreted using one of these prepropeptide portions varied by 1 to 4 additional N-terminal amino acid residues derived from the junction sequence between the sequence for propeptide portion and mature hGH or from C-terminal region of the propeptide portion. These results suggest that the prepropeptide portion can be generally processed even in the heterogeneous fusion. A probable mechanism of processing and maturation of the fusion gene products is also discussed.     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis.

The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by penI and penJ is discussed.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus.

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.